Inhibitors of p38

ABSTRACT

The present invention relates to inhibitors of p38, a mammalian protein kinase involved cell proliferation, cell death and response to extracellular stimuli. The invention also relates to methods for producing these inhibitors. The invention also provides pharmaceutical compositions comprising the inhibitors of the invention and methods of utilizing those compositions in the treatment and prevention of various disorders.

This application claims the benefit of U.S. Provisional Application No.60/034,288 filed Dec. 18, 1996.

TECHNICAL FIELD OF INVENTION

The present invention relates to inhibitors of p38, a mammalian proteinkinase involved cell proliferation, cell death and response toextracellular stimuli. The invention also relates to methods forproducing these inhibitors. The invention also provides pharmaceuticalcompositions comprising the inhibitors of the invention and methods ofutilizing those compositions in the treatment and prevention of variousdisorders.

BACKGROUND OF THE INVENTION

Protein kinases are involved in various cellular responses toextracellular signals. Recently, a family of mitogen-activated proteinkinases (MAPK) have been discovered. Members of this family are Ser/Thrkinases that activate their substrates by phosphorylation B. Stein etal., Ann. Rep. Med. Chem., 31, pp. 289-98 (1996)!. MAPKs are themselvesactivated by a variety of signals including growth factors, cytokines,UV radiation, and stress-inducing agents.

One particularly interesting MAPK is p38. p38, also known as cytokinesuppressive anti-inflammatory drug binding protein (CSBP) and RK, wasisolated from murine pre-B cells that were transfected with thelipopolysaccharide (LPS) receptor CD14 and induced with LPS. p38 hassince been isolated and sequenced, as has the cDNA encoding it in humansand mouse. Activation of p38 has been observed in cells stimulated bystresses, such as treatment of lipopolysaccharides (LPS), UV,anisomycin, or osmotic shock, and by cytokines, such as IL-1 and TNF.

Inhibition of p38 kinase leads to a blockade on the production of bothIL-1 and TNF. IL-1 and TNF stimulate the production of otherproinflammatory cytokines such as IL-6 and IL-8 and have been implicatedin acute and chronic inflammatory diseases and in post-menopausalosteoporosis R. B. Kimble et al., Endocrinol., 136, pp. 3054-61 (1995)!.

Based upon this finding it is believed that p38, along with other MAPKs,have a role in mediating cellular response to inflammatory stimuli, suchas leukocyte accumulation, macrophage/monocyte activation, tissueresorption, fever, acute phase responses and neutrophilia. In addition,MAPKs, such as p38, have been implicated in cancer, thrombin-inducedplatelet aggregation, immunodeficiency disorders, autoimmune diseases,cell death, allergies, osteoporosis and neurodegenerative disorders.Inhibitors of p38 have also been implicated in the area of painmanagement through inhibition of prostaglandin endoperoxide synthase-2induction. Other diseases associated with Il-1, IL-6, IL-8 or TNFoverproduction are set forth in WO 96/21654.

Others have already begun trying to develop drugs that specificallyinhibit MAPKs. For example, PCT publication WO 95/31451 describespyrazole compounds that inhibit MAPKs, and in particular p38. However,the efficacy of these inhibitors in vivo is still being investigated.

Accordingly, there is still a great need to develop other potent,p38-specific inhibitors that are useful in treating various conditionsassociated with p38 activation.

SUMMARY OF THE INVENTION

The present invention solves this problem by providing compounds whichdemonstrate strong and specific inhibition of p38.

These compounds have the general formula: ##STR1## wherein each of Q₁and Q₂ are independently selected from 5-6 membered aromatic carbocyclicor heterocyclic ring systems, or 8-10 membered bicyclic ring systemscomprising aromatic carbocyclic rings, aromatic heterocyclic rings or acombination of an aromatic carbocyclic ring and an aromatic heterocyclicring.

The rings that make up Q₁ are substituted with 1 to 4 substituents, eachof which is independently selected from halo; C₁ -C₃ alkyl; C₁ -C₃ alkylsubstituted with NR', OR', CO₂ R' or CONR'₂ ; O--(C₁ -C₃)-alkyl; O--(C₁-C₃)-alkyl substituted with NR', OR', CO₂ R' or CONR'₂ ; NR'₂ ; OCF₃ ;CF₃ ; NO₂ ; CO₂ R'; CONR'; SR'; S(O₂)N(R')₂ ; SCF₃ ; or CN. The ringsthat make up Q₂ are optionally substituted with up to 4 substituents,each of which is independently selected from halo; C₁ -C₃ alkyl; C₁ -C₃alkyl substituted with NR', OR', CO₂ R' or CONR'₂ ; O--(C₁ -C₃)-alkyl;O--(C₁ -C₃)-alkyl substituted with NR', OR', CO₂ R' or CONR'₂ ; NR'₂ ;OCF₃ ; CF₃ ; NO₂ ; CO₂ R'; CONR'; SR'; S(O₂) N (R')₂ ; SCF₃ ; or CN.

R' is selected from hydrogen, (C₁ -C₃)-alkyl or (C₂ -C₃)-alkenyl oralkynyl.

X is selected from --S--, --O--, --S(O₂)--, --S(O)--, --S(O₂)--N(R)--,--N(R)--S(O₂)--, --N(R)--C(O)O--, --O--C(O)--N(R)--, --C(O)--,--C(O)O--, --O--C(O)--, --C(O)--N(R)--, --N(R)--C(O)--, --N(R)--, or--C(R₂)--.

Each R is independently selected from hydrogen, --R², --NR² ₂, --OR²,SR², --C(O)--NR² ₂ or --C(O)--OR² wherein two adjacent R are optionallybound to one another and, together with each Y to which they arerespectively bound, form a 4-8 membered carbocyclic or heterocyclicring;

R² is selected from hydrogen, (C₁ -C₃)-alkyl, O--(C₂ -C₃)-alkyl, C₁-C₃)-alkenyl or O--(C₂ -C₃)-alkenyl each optionally substituted with W.

Y is N or C;

A, if present, is N or CR';

n is 0 or 1;

R₁ is selected from hydrogen, (C₁ -C₃)-alkyl, OH, or O--(C₁ -C₃)-alkyl.

W is selected from ##STR2## --N(R')₂, --OR', --SR', --S(O)R', --S(O₂)R',C(O)OR' or any 5 to 7-membered heterocyclic ring comprising up to 3heteroatoms independently selected from N, O or S, wherein saidheterocyclic ring is optionally substituted with up to 3 substituentsindependently selected from halo, C₁ -C₃ alkyl, O--(C₁ -C₃)-alkyl, NR'₂,OCF₃, CF₃, NO₂, CO₂ R', CONR' or CN.

In another embodiment, the invention provides pharmaceuticalcompositions comprising the p38 inhibitors of this invention. Thesecompositions may be utilized in methods for treating or preventing avariety of disorders, such as cancer, inflammatory diseases, autoimmunediseases, destructive bone disorders, proliferative disorders,infectious diseases, viral diseases and neurodegenerative diseases.These compositions are also useful in methods for preventing cell deathand hyperplasia and therefore may be used to treat or preventreperfusion/ischemia in stroke, heart attacks, organ hypoxia. Thecompositions are also useful in methods for preventing thrombin-inducedplatelet aggregation. Each of these above-described methods is also partof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides inhibitors of p38 having the generalformula: ##STR3## wherein each of Q₁ and Q₂ are independently selectedfrom 5-6 membered aromatic carbocyclic or heterocyclic ring systems, or8-10 membered bicyclic ring systems comprising aromatic carbocyclicrings, aromatic heterocyclic rings or a combination of an aromaticcarbocyclic ring and an aromatic heterocyclic ring.

The rings that make up Q₁ are substitued with 1 to 4 substituents, eachof which is independently selected from halo; C₁ -C₃ alkyl; C₁ -C₃ alkylsubstituted with NR', OR', CO₂ R' or CONR'₂ ; O--(C₁ -C₃)-alkyl; O--(C₁-C₃)-alkyl substituted with NR', OR', CO₂ R' or CONR'₂ ; NR'₂ ; OCF₃ ;CF₃ ; NO₂ ; CO₂ R'; CONR'; SR'; S(O₂)N(R')₂ ; SCF₃ ; or CN. The ringsthat make up Q₂ are optionally substituted with up to 4 substituents,each of which is independently selected from halo; C₁ -C₃ alkyl; C₁ -C₃alkyl substituted with NR', OR', CO₂ R' or CONR'₂ ; O--(C₁ -C₃)-alkyl;O--(C₁ -C₃)-alkyl substituted with NR', OR', CO₂ R' or CONR'₂ ; NR'₂ ;OCF₃ ; CF₃ ; NO₂ ; CO₂ R'; CONR'; SR'; S(O₂)N(R') ₂ ; SCF₃ ; or CN.

R' is selected from hydrogen, (C₁ -C₃)-alkyl or (C₂ -C₃)-alkenyl oralkynyl.

X is selected from --S--, --O--, --S(O₂)--, --S(O)--, --S(O₂)--N(R)--,--N(R)--S(O₂)--, --N(R)--C(O)O--, --O--C(O)--N(R)--, --C(O)--,--C(O)O--, --O--C(O)--, --C(O)--N(R)--, --N(R)--C(O)--, --N(R)--, or--C(R₂)--.

Each R is independently selected from hydrogen, --R², --NR² ₂, --OR²,SR², --C(O)--NR² or --C(O)--OR wherein two adjacent R are optionallybound to one another and, together with each Y to which they arerespectively bound, form a 4-8 membered carbocyclic or heterocyclicring;

When the two R components form a ring together with the Y components towhich they are respectively bound, it will obvious to those skilled inthe art that a terminal hydrogen from each unfused R component will belost. For example, if a ring structure is formed by binding those two Rcomponents together, one being --NH--CH₃ and the other being --CH₂--CH₃, one terminal hydrogen on each R component (indicated in bold)will be lost. Therefore, the resulting portion of the ring structurewill have the formula --NH--CH₂ --CH₂ --CH₂ --.

R² is selected from hydrogen, (C₁ -C₃)-alkyl, O--(C₂ -C₃)-alkyl, C₁-C₃)-alkenyl or O--(C₂ -C₃)-alkenyl each optionally substituted with W.

Y is N or C;

A, if present, is N or CR';

n is 0 or 1;

R₁ is selected from hydrogen, (C₁ -C₃)-alkyl, OH, or O--(C₁ -C₃)-alkyl.It will be apparent to those of skill in the art that if R₁ is OH, theresulting inhibitor may tautomerize resulting in a compound of theformula: ##STR4## which are also p38 inhibitors of this invention.

W is selected from ##STR5## --N(R')₂, --OR', --SR', --S(O)R', --S(O₂)R',C(O)OR' or any 5 to 7-membered heterocyclic ring comprising up to 3heteroatoms independently selected from N, O or S, wherein saidheterocyclic ring is optionally substituted with up to 3 substituentsindependently selected from halo, C₁ -C₃ alkyl, O--(C₁ -C₃)-alkyl, NR'₂,OCF₃, CF₃, NO₂, CO₂ R', CONR' or CN.

According to another preferred embodiment, Q₁ is selected from phenyl orpyridyl containing 1 to 3 substituents, wherein at least one of saidsubstituents is in the ortho position and said substituents areindependently selected from chloro, fluoro, bromo, --CH₃, --OCH₃, --OH,--CF₃, --OCF₃, --O(CH₂)₂ CH₃ or NH₂. Even more preferred are phenyl orpyridyl containing at least 2 of the above-indicated substituents bothbeing in the ortho position.

Some specific examples of preferred Q₁ are: ##STR6##

Most preferably, Q₁ is selected from 2,6-difluorophenyl or2,6-dichlorophenyl.

According to a preferred embodiment, Q₂ is phenyl or pyridyl containing0 to 3 substituents, wherein each substituent is independently selectedfrom chloro, fluoro, methyl, --OCH₃, --OH, --NH₂, --CF₃, --OCF₃, --SCH₃,--OCH₃, --C(O)OH and --CH₂ CH₂ OH.

Some specific examples of preferred Q₂ are: ##STR7## or unsubstitutedphenyl. Most preferred are compounds wherein Q₂ is selected from phenyland 4-fluorophenyl.

According to yet another preferred embodiment, X is --S--, --O--,--S(O₂)--, --S(O)--, --NR--, --C(R₂)-- or --C(O)--. Most preferably, Xis S.

According to another preferred embodiment, n is 1 and A is N.

According to another preferred embodiment, each Y is C.

According an even more preferred embodiment, each Y is C and the Rattached to those Y components is selected from hydrogen or methyl.

Some specific inhibitors of this invention are set forth in the tablebelow.

                  TABLE 1                                                         ______________________________________                                        1 #STR8##                                                                     Cmpd #                                                                              Q.sub.1      Q.sub.2     R.sub.a                                                                              R.sub.b                                 ______________________________________                                         2    4-fluorophenyl                                                                             phenyl      hydrogen                                                                             hydrogen                                 3    2,4-dichlorophenyl                                                                         phenyl      hydrogen                                                                             hydrogen                                 5    2,4-dichlorophenyl                                                                         4-methylphenyl                                                                            hydrogen                                                                             hydrogen                                 6    2,6-dichlorophenyl                                                                         phenyl      hydrogen                                                                             hydrogen                                 7    2-chlorophenyl                                                                             phenyl      hydrogen                                                                             hydrogen                                 8    2-methylphenyl                                                                             phenyl      hydrogen                                                                             hydrogen                                 9    3,4-dichlorophenyl                                                                         phenyl      hydrogen                                                                             hydrogen                                10    4-methoxyphenyl                                                                            phenyl      hydrogen                                                                             hydrogen                                11    2-methoxyphenyl                                                                            phenyl      hydrogen                                                                             hydrogen                                12    2,6-dichlorophenyl                                                                         4-fluorophenyl                                                                            hydrogen                                                                             hydrogen                                13    2,6-dichlorophenyl                                                                         phenyl      methyl methyl                                  14    2,6-dichlorophenyl                                                                         4-methylphenyl                                                                            hydrogen                                                                             hydrogen                                15    2,6-dichlorophenyl                                                                         3-methylphenyl                                                                            hydrogen                                                                             hydrogen                                16    2,6-dichlorophenyl                                                                         3,4-        hydrogen                                                                             hydrogen.                                                  dichlorophenyl                                             ______________________________________                                    

According to another embodiment, the present invention provides methodsof producing inhibitors of p38 of the formula (Ia) depicted above. Thesemethods involve reacting a compound of formula II: ##STR9## wherein eachof the variables in the above formula are the same as defined above forthe inhibitors of this invention, with a leaving group reagent offormula IIa: ##STR10## wherein R' is as defined above, or a leavinggroup reagent of formula IIb: ##STR11## wherein each of L₁, L₂, and L₃independently represents a leaving group.

The leaving group reagent used in this reaction is added in excess,either neat or with a co-solvent, such as toluene. The reaction iscarried out at a temperature of between 25° C. and 150° C.

Leaving group reagents of formula IIa that are useful in producing thep38 inhibitors of this invention include dimethylformamidedimethylacetal, dimethylacetamide dimethylacetal, trimethylorthoformate, dimethylformamide diethylacetal and other relatedreagents. Preferably the leaving group reagents of formula IIa used toproduce the inhibitors of this invention is dimethylformamidedimethylacetal.

Leaving group reagents of formula IIb that are useful in producing thep38 inhibitors of this invention include phosgene, carbonyldiamidazoleand triphosgene.

More preferred methods of producing the compounds of this inventionutilize compounds of formula II wherein each of the variables aredefined in terms of the more preferred and most preferred choices as setforth above for the compounds of this invention.

Because the source of R₁ is the leaving group reagent (C--R' or C═O),its identity is, of course, dependent on the structure of that reagent.Therefore, in compounds where R' is OH, the reagent used must be IIb.Similarly, when R' is H or (C₁ -C₃)-alkyl, the reagent used must be IIa.In order to generate inhibitors wherein R₁ is O--(C₁ -C₃)-alkyl, acompound wherein R₁ is OH is first generated, followed by alkylation ofthat hydroxy by standard techniques, such as treatment with Na hydridein DMF, methyl iodide and ethyl iodide.

The immediate precursors to the inhibitors of this invention of formulaIa (i.e., compounds of Formula II) may themselves be synthesized byeither of the synthesis schemes depicted below: ##STR12##

In Scheme 1, the order of steps 1) and 2) can be reversed. Also, thestarting nitrile may be replaced by a corresponding acid or by an ester.Alternatively, other well-known latent carboxyl or carboxamide moietiesmay be used in place of the nitrile (see scheme 2). Variations such ascarboxylic acids, carboxylic esters, oxazolines or oxizolidinones may beincorporated into this scheme by utilizing subsequent deprotection andfuntionalization methods which are well known in the art

The base used in the first step of Scheme 1 (and in Scheme 2, below) isselected from sodium hydride, sodium amide, LDA, lithiumhexamethyldisilazide, sodium hexamethyldisilazide or any number of othernon-nucleophilic bases that will deprotonate the position alpha to thenitrile.

Also, the addition of HX-Q₂ in the single step depicted above may besubstituted by two steps--the addition of a protected or unprotected Xderivative followed by the addition of a Q₂ derivative in a subsequentstep. ##STR13##

In Scheme 2, Z is selected from COOH, COOR, CON(R₂), oxazoline,oxazolidinone or CN. R is as defined above.

According to another embodiment, the present invention provides methodsof producing inhibitors of p38 of the formula (Ib) depicted above. Thesemethods involve reacting a compound of formula III: ##STR14## whereineach of the variables in the above formula are the same as defined abovefor the inhibitors of this invention, with a leaving group reagent offormula: ##STR15## as described above.

Two full synthesis schemes for the p38 inhibitors of formula (Ib) ofthis invention are depicted below. ##STR16##

In scheme 3, a Q1 substituted derivative may be treated with a base suchas sodium hydride, sodium amide, LDA, lithium hexamethyldisilazide,sodium hexamethyldisilazide or any number of other non-nucleophilicbases to deprotonate the position alpha to the Z group, which representsa masked amide moiety. Alternatively, Z is a carboxylic acid, carboxylicester, oxazoline or oxizolidinone. The anion resulting fromdeprotonation is then contacted with a nitrogen bearing heterocycliccompound which contains two leaving groups, or latent leaving groups.One example of such compound may be 2,6-dichloropyridine.

In step two, the Q2 ring moiety is introduced. This may be performedutilizing many reactions well known in the art which result in theproduction of biaryl compounds. One example may be the reaction of anaryl lithium compound with the pyridine intermediate produced in step 1.Alternatively, an arylmetallic compound such as an aryl stannane may bereacted with the aryl halide portion of the pyridine intermediate in thepresence of a Pd° catalyst.

In step 3 the Z group is deprotected and/or functionalized to form theamide compound. When Z is a carboxylic acid, carboxylic ester, oxazolineor oxizolidinone, variations in deprotection and funtionalizationmethods which are well known in the art are employed to produce theamide. Finally in step 4, the amide compound is cyclized to the finalproduct utilizing reagants such as DMF acetal or similar reagents eitherneat or in an organic solvent. ##STR17##

Scheme 4 is similar except that the a biaryl intermediate is firstgenerated prior to reaction with the Q1 starting material.

The activity of the p38 inhibitors of this invention may be assayed byin vitro, in vivo or in a cell line. In vitro assays include assays thatdetermine inhibition of either the kinase activity or ATPase activity ofactivated p38. Alternate in vitro assays quantitate the ability of theinhibitor to bind to p38 and may be measured either by radiolabellingthe inhibitor prior to binding, isolating the inhibitor/p38 complex anddetermining the amount of radiolabel bound, or by running a competitionexperiment where new inhibitors are incubated with p38 bound to knownradioligands.

Cell culture assays of the inhibitory effect of the compounds of thisinvention may determine the amounts of TNF, IL-1, IL-6 or IL-8 producedin whole blood or cell fractions thereof in cells treated with inhibitoras compared to cells treated with negative controls. Level of thesecytokines may be determined through the use of commercially availableELISAs.

An in vivo assay useful for determining the inhibitory activity of thep38 inhibitors of this invention are the suppression of hindpaw edema inrats with Mycobacterium butyricum-induced adjuvant arthritis. This isdescribed in J. C. Boehm et al., J. Med. Chem., 39, pp. 3929-37 (1996),the disclosure of which is herein incorporated by reference. The p38inhibitors of this invention may also be assayed in animal models ofarthritis, bone resorption, endotocin shock and immune function, asdescribed in A. M. Badger et al., J. Pharmacol. ExperimentalTherapuetics, 279, pp. 1453-61 (1996), the disclosure of which is hereinincorporated by reference.

The p38 inhibitors or pharmaceutical salts thereof may be formulatedinto pharmaceutical compositions for administration to animals orhumans. These pharmaceutical compositions, which comprise and amount ofp38 inhibitor effective to treat or prevent a p38-mediated condition anda pharmaceutically acceptable carrier, are another embodiment of thepresent invention.

The term "p38-mediated condition", as used herein means any disease orother deleterious condition in which p38 is known to play a role. Thisincludes, conditions which are known to be caused by IL-1, TNF, IL-6 orIL-8 overproduction. Such conditions include, without limitation,inflammatory diseases, autoimmune diseases, destructive bone disorders,proliferative disorders, infectious diseases, viral disease, andneurodegenerative diseases.

Inflammatory diseases which may be treated or prevented include, but arenot limited to acute pancreatitis, chronic pancreatitis, asthma,allergies, and adult respiratory distress syndrome.

Autoimmune diseases which may be treated or prevented include, but arenot limited to, glomeralonephritis, rheumatoid arthritis, systemic lupuserythematosus, scleroderma, chronic thyroiditis, Graves' disease,autoimmune gastritis, insulin-dependent diabetes mellitus (Type I),autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia,atopic dermatitis, chronic active hepatitis, myasthenia gravis, multiplesclerosis, inflammatory bowel disease, ulcerative colitis, Crohn'sdisease, psoriasis, or graft vs. host disease.

Destructive bone disorders which may be treated or prevented include,but are not limited to, osteoporosis, osteoarthritis and multiplemyeloma-related bone disorder.

Proliferative diseases which may be treated or prevented include, butare not limited to, acute myelogenous leukemia, chronic myelogenousleukemia, metastatic melanoma, Kaposi's sarcoma, and multiple myeloma.

Infectious diseases which may be treated or prevented include, but arenot limited to, sepsis, septic shock, and Shigellosis.

Viral diseases which may be treated or prevented include, but are notlimited to, acute hepatitis infection (including hepatitis A, hepatitisB and hepatitis C), HIV infection and CMV retinitis.

Degenerative or diseases which may be treated or prevented by thecompounds of this invention include, but are not limited to, Alzheimer'sdisease, Parkinson's disease, cerebral ischemia, and otherneurodegenerative diseases.

"p38-mediated conditions" also include ischemia/reperfusion in stroke,heart attacks, myocardial ischemia, organ hypoxia, vascular hyperplasia,cardiac hypertrophy, and thrombin-induced platelet aggregation.

In addition, p38 inhibitors in this invention are also capable ofinhibiting the expression of inducible pro-inflammatory proteins such asprostaglandin endoperoxide synthase-2 (PGHS-2), also referred to ascyclooxygenase-2 (COX-2). Therefore, other "p38-mediated conditions" areedema, analgesia, fever and pain, such as neuromuscular pain, headache,cancer pain, dental pain and arthritis pain.

The diseases that may be treated or prevented by the p38 inhibitors ofthis invention may also be conveniently grouped by the cytokine (IL-1,TNF, IL-6, IL-8) that is believed to be responsible for the disease.

Thus, an IL-1-mediated disease or condition includes rheumatoidarthritis, osteoarthritis, stroke, endotoxemia and/or toxic shocksyndrome, inflammatory reaction induced by endotoxin, inflammatory boweldisease, tuberculosis, atherosclerosis, muscel degeneration, cachexia,psoriatic arthritis, Reiter's syndrome, gout, traumatic arthritis,rubella arthritis, acute synovitis, diabetes, pancreatic B-cell diseaseand Alzheimer's disease.

TNF-mediated disease or condition includes, rheumatoid arthritis,rheumatoid spndylitis, osteoarthritis, gouty arthritis and otherarthritic conditions, sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, adult respiratory distresssyndrome, cerebral malaria, chronic pulmonary inflammatory disease,silicosis, pulmonary sarcoisosis, bone resorption diseases, reperfusioninjury, graft vs. host reaction, allograft rejections, fever andmyalgias due to infection, cachexia secondary to infection, AIDS, ARC ormalignancy, keloid formation, scar tissue formation, Crohn's disease,ulcerative colitis or pyresis. TNF-mediated diseases also include viralinfections, such as HIV, CMV, influenza and herpes; and vetinary viralinfections, such as lentivirus infections, including, but not limited toequine infectious anaemia virus, caprine arthritis virus, visna virus ormaedi virus; or retrovirus infections, including feline immunodeficiencyvirus, bovine immunodeficiency virus, or canine immunodeficiency virus.

IL-8 mediated disease or conditon includes diseases characterized bymassive neutrophil infiltration, such as psoriasis, inflammatory boweldisease, asthma, cardiac and renal reperfusion injury, adult respiratorydistress syndrome, thrombosis and glomerulonephritis.

In addition, the compounds of this infection may be used topically totreat or prevent conditions caused or exacerbated by IL-1 or TNF. Suchconditions include inflamed joints, eczema, psoriasis, inflammatory skinconditions such as sunburn, inflammatory eye conditions such asconjuctivitis, pyresis, pain and other conditions associated withinflammation.

Pharmaceutically acceptable carriers that may be used in thesepharmaceutical compositions include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term "parenteral"as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient which is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions can be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of p38 inhibitor that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated, the particular mode of administration. Preferably, thecompositions should be formulated so that a dosage of between 0.01-100mg/kg body weight/day of the inhibitor can be administered to a patientreceiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of inhibitor will also depend upon the particular compound in thecomposition.

According to another embodiment, the invention provides methods fortreating or preventing a p38-mediated condition comprising the step ofadministering to a patient one of the above-described pharmaceuticalcompositions. The term "patient", as used herein, means an animal,preferably a human.

Preferably, that method is used to treat or prevent a condition selectedfrom inflammatory diseases, autoimmune diseases, destructive bonedisorders, proliferative disorders, infectious diseases, degenerativediseases, allergies, reperfusion/ischemia in stroke, heart attacks,organ hypoxia, vascular hyperplasia, cardiac hypertrophy, andthrombin-induced platelet aggregation.

According to another embodiment, the inhibitors of this invention areused to treat or prevent an IL-1, IL-6, IL-8 or TNF-mediated disease orcondition. Such conditions are described above.

Depending upon the particular p38-mediated condition to be treated orprevented, additional drugs, which are normally administered to treat orprevent that condition may be administered together with the inhibitorsof this invention. Those additional agents may be administeredseparately, as part of a multiple dosage regimen, from the p38inhibitor-containing composition. Alternatively, those agents may bepart of a single dosage form, mixed together with the p38 inhibitor in asingle composition.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLE 1 Synthesis of p38 Inhibitor Compound 1

Examples of the synthesis of several compounds of formula Ia are setforth in the following 4 examples. ##STR18##

To a slurry of sodium amide, 90% (1.17 g., 30 mmol) in drytetrahydrofuran (20 ml) we added a solution of benzyl cyanide (2.92 g.,25.0 mmol) in dry tetrahydrofuran (10 ml) at room temperature. Themixture was stirred at room temperature for 30 minutes. To the reactionmixture we added a solution of 3,6-dichloropyridazine (3.70 g., 25.0mmol) in dry tetrahydrofuran (10 ml). After stirring for 30 minutes, thereaction mixture was diluted with an aqueous saturated sodiumbicarbonate solution. The reaction mixture was then extracted with ethylacetate. The layers were separated and the organic was washed withwater, brine, dried over magnesium sulfate, filtered and concentrated invacuo.

The residue was purified by chromatography on silica gel (eluant: 30%ethyl acetate in n-hexane) to give 3.71 g. (16.20 mmol˜54%) of productas a white solid. ##STR19##

To a slurry of sodium hydride, 95% (0.14 g., 6.0 mmol) in drytetrahydrofuran (10 ml) we added thiophenol (0.66g, 6.0 ml.) at roomtemperature. The reaction mixture was then stirred for 10 minutes. Tothe reaction mixture we added a solution of the product from step A.,above (1.31 g., 5.72 mmol) in absolute ethanol (20 ml.). The reactionmixture was then brought to reflux and stirred there for one hour. Thecool reaction mixture was concentrated in vacuo. The residue was dilutedwith a 1N sodium hydroxide solution (10 ml), then extracted withmethylene chloride. The organic phase was washed with water, brine,dried over magnesium sulfate and concentrated in vacuo.

The residue was purified by chromatography on silica gel (eluant: 20%ethyl acetate in n-hexane) to give 0.66g. (2.19 mmol˜40%) of product asa white solid. ##STR20##

A mixture of the product from step B. (0.17 g., 0.69 mmol) andconcentrated sulfuric acid (5 ml) was heated to 100° C. for one hour.The solution was cooled and adjusted to pH 8 with a saturated sodiumbicarbonate solution. The reaction mixture was extracted with methylenechloride. The organic layer was washed with water, brine, dried overmagnesium sulfate and concentrated in vacuo to give 0.22 g. (0.69mmol˜100%) of compound pre-1 as an orange oil. ¹ H NMR (500 MHz, CD3OD)d7.7 (d), 7.5 (d), 7.4 (m) 7.3-7.2 (m). ##STR21##

A solution of pre-1 from step C. (0.22 g., 0.69 mmol) andN,N-dimethylformamide dimethylacetal (0.18 g., 1.5 mmol) in toluene (5ml) was heated at 100° C. for one hour. Upon cooling, the resultingsolid was filtered and dissolved in warm ethyl acetate. The product wasprecipitated with the dropwise addition of diethyl ether. The productwas then filtered and washed with diethyl ether to give 0.038 g. ofcompound 1 (which is depicted in Table 1) as a yellow solid. ¹ H NMR(500 MHz, CDCl3) d8.63 (s), 7.63-7.21 (m), 6.44 (d).

EXAMPLE 2 Synthesis of p38 Inhibitor Compound 2 ##STR22##

The first intermediate depicted above was prepared in a similar manneras in Example 1A, using 4-fluorophenylacetonitrile, to afford 1.4 g (5.7mmol, ˜15%) of product. ##STR23##

The above intermediate was prepared in a similar manner as in Example1B. This afforded 0.49 g (1.5 mmol, 56%) of product. ##STR24##

The above intermediate was prepared in a similar manner as Example 1C.This afforded 0.10 g (0.29 mmol, 45%) of compound pre-2. ¹ H NMR (500MHz, CDCl3) d 7.65-7.48 (m), 7.47-7.30 (m), 7.29-7.11 (m), 7.06-6.91(m), 5.85 (s, br). ##STR25##

Compound 2 (which is depicted in Table 1) was prepared from pre-2 in asimilar manner as in Example 1D. This afforded 0.066 g of product. ¹ HNMR (500 MHz, CDCl3) d 8.60 (s), 7.62-7.03 (m), 6.44 (d)).

EXAMPLE 3 Synthesis of p38 Inhibitor Compound 6 ##STR26##

The first intermediate in the preparation of compound 6 was prepared ina manner similar to that described in Example 1A, using2,6-dichlorophenylacetonitrile, to afford 2.49 g (8.38, 28%) of product.##STR27##

The next step in the synthesis of compound 6 was carried out in asimilar manner as described in Example 1B. This afforded 2.82 g (7.6mmol, 91%) of product. ##STR28##

The final intermediate, pre-6, was prepared in a similar manner asdescribed in Example 1C. This afforded 0.89 g (2.3 mmol, 85%) ofproduct. ¹ H NMR (500 MHz, CD3OD) d 7.5-7.4 (dd), 7.4 (m), 7.3 (d), 7.2(m), 7.05 (d). ##STR29##

The final step in the synthesis of compound 6 (which is depicted inTable 1) was carried out as described in Example 1D. This afforded 0.06g of product. ¹ H NMR (500 MHz, CDCl3) d 8.69 (s), 7.65-7.59 (d),7.58-7.36 (m), 7.32-7.22 (m), 6.79 (d), 6.53 (d).

EXAMPLE 4 Preparation of p38 Inhibitor Compound 5 ##STR30##

The first intermediate in the synthesis of compound 5 was prepared in asimilar manner as described in Example 1A, using2,4-dichlorophenylacetonitrile, to afford 3.67 g (12.36 mmol, 49%) ofproduct. ##STR31##

The second intermediate was prepared in a similar manner as described inExample 1B. This afforded 3.82 g (9.92 mmol, 92%) of product. ##STR32##

The final intermediate, pre-5, was prepared in a similar manner asdescribed in Example 1C. This afforded 0.10 g (0.24 mmol, 92%) ofproduct. ¹ H NMR (500 MHz, CD3OD) d 7.9 (d), 7.7 (d), 7.6-7.5 (dd),7.4-7.3 (m), 2.4 (s). ##STR33##

The final step in the preparation of compound 5 (which is depicted inTable 1) was carried out in a similar manner as described in Example 1D.This afforded 0.06 g of product. ¹ H NMR (500 MHz, CDCl3) d 8.64 (s),7.51-7.42 (m), 7.32-7.21 (m), 6.85 (d), 6.51 (d), 2.42 (s).

Other compounds of formula Ia of this invention may be synthesized in asimilar manner using the appropriate starting materials.

EXAMPLE 5 Preparation of p38 Inhibitor Compound 14

An example of the synthesis of a p38 inhibitor of this invention of theformula Ib is presented below. ##STR34##

To a slurry of sodium amide, 90% (1.1 eq) in dry tetrahydrofuran isadded a solution of 2,6-dichlorobenzyl cyanide (1.0 eq) in drytetrahydrofuran at room temperature. The mixture is stirred at roomtemperature for 30 minutes. To the reaction mixture is added a solutionof 2,6-dichloropyridine (1 eq) in dry tetrahydrofuran. The reaction ismonitored by TLC and, when complete, the reaction mixture is dilutedwith an aqueous saturated sodium bicarbonate solution. The reactionmixture is then extracted with ethyl acetate. The layers are separatedand the organic layer is washed with water, brine, dried over magnesiumsulfate, filtered and concentrated in vacuo. The residue is purified bychromatography on silica gel to yield pure product. ##STR35##

To a solution of 4-fluoro-bromobenzene (1 eq) in dry tetrahydrofuran at-78° C. is added t-butyllithium (2 eq, solution in hexanes). Thereaction mixture is then stirred for 30 minutes. To the reaction mixtureis added a solution of the product from Step A (1 eq) in dry THF. Thereaction mixture is then monitored and slowly brought to roomtemperature. The reaction mixture is quenched with water then extractedwith methylene chloride. The organic phase is washed with water, brine,dried over magnesium sulfate and concentrated in vacuo. The residue ispurified by chromatography on silica gel to yield the product. ##STR36##

A mixture of the product step B and concentrated sulfuric acid is heatedto 100° C. for one hour. The solution is cooled and adjusted to pH 8with a saturated sodium bicarbonate solution. The reaction mixture isextracted with methylene chloride. The organic layer is washed withwater, brine, dried over magnesium sulfate and concentrated in vacuo togive product. The final product is purified by silica gel flashchromatography ##STR37##

A solution of the product Step C (1 eq) and N,N-Dimethylformamidedimethylacetal (2 eq) in toluene is heated at 100° C. for one hour. Uponcooling, the resulting mixture is filtered and dissolved in warm ethylacetate. The product is precipitated with the dropwise addition ofdiethyl ether. The product is then filtered and washed with diethylether to give Compound 14. The final product is further purified bysilica gel chromatography.

Other compounds of formula Ib of this invention may be synthesized in asimilar manner using the appropriate starting materials.

EXAMPLE 6 Cloning of p38 Kinase in Insect Cells

Two splice variants of human p38 kinase, CSBP1 and CSBP2, have beenidentified. Specific oligonucleotide primers were used to amplify thecoding region of CSBP2 cDNA using a HeLa cell library (Stratagene) as atemplate. The polymerase chain reaction product was cloned into thepET-15b vector (Novagen). The baculovirus transfer vector, pVL-(His)₆-p38 was constructed by subcloning a XbaI-BamHI fragment ofpET15b-(His)₆ -p38 into the complementary sites in plasmid pVL1392(Pharmingen).

The plasmid pVL-(His)₆ -p38 directed the synthesis of a recombinantprotein consisting of a 23-residue peptide (MGSSHHHHHHSSGLVPRGSHMLE,where LVPRGS represents a thrombin cleavage site) fused in frame to theN-terminus of p38, as confirmed by DNA sequencing and by N-terminalsequencing of the expressed protein. Monolayer culture of Spodopterafrugiperda (Sf9) insect cells (ATCC) was maintained in TNM-FH medium(Gibco BRL) supplemented with 10% fetal bovine serum in a T-flask at 27°C. Sf9 cells in log phase were co-transfected with linear viral DNA ofAutographa califonica nuclear polyhedrosis virus (Pharmingen) andtransfer vector pVL-(His)₆ -p38 using Lipofectin (Invitrogen). Theindividual recombinant baculovirus clones were purified by plaque assayusing 1% low melting agarose.

EXAMPLE 7 Expression And Purification of Recombinant p38 Kinase

Trichoplusia ni (Tn-368) High-Five™ cells (Invitrogen) were grown insuspension in Excel-405 protein free medium (JRH Bioscience) in a shakerflask at 27° C. Cells at a density of 1.5×10⁶ cells/ml were infectedwith the recombinant baculovirus described above at a multiplicity ofinfection of 5. The expression level of recombinant p38 was monitored byimmunoblotting using a rabbit anti-p38 antibody (Santa CruzBiotechnology). The cell mass was harvested 72 hours after infectionwhen the expression level of p38 reached its maximum.

Frozen cell paste from cells expressing the (His)₆ -tagged p38 wasthawed in 5 volumes of Buffer A (50 mM NaH₂ PO₄ pH 8.0, 200 mM NaCl, 2mMβ-Mercaptoethanol, 10% Glycerol and 0.2 mM PMSF). After mechanicaldisruption of the cells in a Microfluidizer, the lysate was centrifugedat 30,000×g for 30 minutes. The supernatant was incubated batchwise for3-5 hours at 4° C. with Talon™ (Clontech) metal affinity resin at aratio of 1 ml of resin per 2-4 mgs of expected p38. The resin wassettled by centrifugation at 500×g for 5 minutes and gently washedbatchwise with Buffer A. The resin was slurried and poured into a column(approx. 2.6×5.0 cm) and washed with Buffer A+5 mM imidizole.

The (His)₆ -p38 was eluted with Buffer A+100 mM imidizole andsubsequently dialyzed overnight at 4° C. against 2 liters of Buffer B,(50 mM HEPES, pH 7.5, 25 mM β-glycerophosphate, 5% glycerol, 2mM DTT).The His₆ tag was removed by addition of at 1.5 units thrombin(Calbiochem) per mg of p38 and incubation at 20° C. for 2-3 hours. Thethrombin was quenched by addition of 0.2 mM PMSF and then the entiresample was loaded onto a 2 ml benzamidine agarose (AmericanInternational Chemical) column.

The flow through fraction was directly loaded onto a 2.6×5.0 cmQ-Sepharose (Pharmacia) column previously equilibrated in Buffer B+0.2mM PMSF. The p38 was eluted with a 20 column volume linear gradient to0.6M NaCl in Buffer B. The eluted protein peak was pooled and dialyzedovernight at 4° C. vs. Buffer C (50 mM HEPES pH 7.5, 5% glycerol, 50 mMNaCl, 2 mM DTT, 0.2 mM PMSF).

The dialyzed protein was concentrated in a Centriprep (Amicon) to 3-4mls and applied to a 2.6×100 cm Sephacryl S-100HR (Pharmacia) column.The protein was eluted at a flow rate of 35 mls/hr. The main peak waspooled, adjusted to 20 mM DTT, concentrated to 10-80 mgs/ml and frozenin aliquots at -70° C. or used immediately.

EXAMPLE 8 Activation of p38

P38 was activated by combining 0.5 mg/ml p38 with 0.005 mg/ml DD-doublemutant MKK6 in Buffer B+10 mM MgCl₂, 2 mM ATP, 0.2 mM Na₂ VO₄ for 30minutes at 20° C. The activation mixture was then loaded onto a 1.0×10cm MonoQ column (Pharmacia) and eluted with a linear 20 column volumegradient to 1.0 M NaCl in Buffer B. The activated p38 eluted after theADP and ATP. The activated p38 peak was pooled and dialyzed againstbuffer B+0.2mM Na₂ VO₄ to remove the NaCl. The dialyzed protein wasadjusted to 1.1M potassium phosphate by addition of a 4.0M stocksolution and loaded onto a 1.0×10 cm HIC (Rainin Hydropore) columnpreviously equilibrated in Buffer D (10% glycerol, 20 mMβ-glycerophosphate, 2.0 mM DTT)+1.1MK₂ HPO₄. The protein was eluted witha 20 column volume linear gradient to Buffer D+50 mM K₂ HPO₄. The doublephosphorylated p38 eluted as the main peak and was pooled for dialysisagainst Buffer B+0.2mM Na₂ VO₄. The activated p38 was stored at -70° C.

EXAMPLE 9 P38 Inhibition Assays

A. Inhibition of Phosphorylation of EGF Receptor Peptide

This assay was carried out in the presence of 10 mM MgCl₂, 25 mMβ-glycerophosphate, 10% glycerol and 100 mM HEPES buffer at pH 7.6. Fora typical IC₅₀ determination, a stock solution was prepared containingall of the above components and activated p38 (5 nM). The stock solutionwas aliquotted into vials. A fixed volume of DMSO or inhibitor in DMSO(final concentration of DMSO in reaction was 5%) was introduced to eachvial, mixed and incubated for 15 minutes at room temperature. EGFreceptor peptide, KRELVEPLTPSGEAPNQALLR, a phosphoryl acceptor inp38-catalyzed kinase reaction (1), was added to each vial to a finalconcentration of 200 μM. The kinase reaction was initiated with ATP (100μM) and the vials were incubated at 30° C. After 30 minutes, thereactions were quenched with equal volume of 10% trifluoroacetic acid(TFA).

The phosphorylated peptide was quantified by HPLC analysis. Separationof phosphorylated peptide from the unphosphorylated peptide was achievedon a reverse phase column (Deltapak, 5 μm, C18 100D, part no. 011795)with a binary gradient of water and acteonitrile, each containing 0.1%TFA. IC₅₀ (concentration of inhibitor yielding 50% inhibition) wasdetermined by plotting the % activity remaining against inhibitorconcentration.

The results for several of the inhibitors of this invention are depictedin Table 2 below:

    ______________________________________                                               Compound                                                                              IC.sub.50 (μM)                                              ______________________________________                                               1       >20                                                                   2       >20                                                                   5       5.0                                                                   6       0.7                                                                   7       4.1                                                                   8       4.4                                                                   9       >20                                                                   10      >20                                                                   12      0.6                                                                   13      1.3                                                                   14      1.4                                                                   15      1.4                                                                   16      0.8                                                            ______________________________________                                    

Pre-2, pre-3 and pre-5 were also tested in this assay. None of thosecompounds demonstrated any inhibition of kinase activity at 20 μM.

Other inhibitors of this invention will also inhibit the kinase activityof p38.

B. Inhibition of ATPase Activity

This assay was carried out in the presence of 10 mM MgCl₂, 25 mmβ-glycerophosphate, 10% glycerol and 100 mM HEPES buffer at pH 7.6. Fora typical K_(i) determination, the K_(m) for ATP in the ATPase activityof activated p38 reaction was determined in the absence of inhibitor andin the presence of two concentrations of inhibitor. A stock solution wasprepared containing all of the above components and activated p38 (60nM). The stock solution was aliquotted into vials. A fixed volume ofDMSO or inhibitor in DMSO (final concentration of DMSO in reaction was2.5%) was introduced to each vial, mixed and incubated for 15 minutes atroom temperature. The reaction was initiated by adding variousconcentrations of ATP and then incubated at 30° C. After 30 minutes, thereactions were quenched with 50 μl of EDTA (0.1 M, final concentration),pH 8.0. The product of p38 ATPase activity, ADP, was quantified by HPLCanalysis.

Separation of ADP from ATP was achieved on a reversed phase column(Supelcosil, LC-18, 3 μm, part no. 5-8985) using a binary solventgradient of following composition: Solvent A--0.1 M phosphate buffercontaining 8 mM tetrabutylammonium hydrogen sulfate (Sigma Chemical Co.,catalogue no. T-7158), Solvent B--Solvent A with 30% methanol.

K_(i) was determined from the rate data as a function of inhibitor andATP concentrations. The results for several of the inhibitors of thisinvention are depicted in Table 3 below:

    ______________________________________                                               Compound                                                                              K.sub.i (μM)                                                ______________________________________                                               1       >20                                                                   2       15                                                                    3       5.0                                                                   5       2.9                                                                   6       0.4                                                            ______________________________________                                    

Other p38 inhibitors of this invention will also inhibit the ATPseactivity of p38.

C. Inhibition of IL-1, TNF, IL-6 and IL-8 Production in LPS-StimulatedPBMCs

Inhibitors were serially diluted in DMSO from a 20 mM stock. At least 6serial dilutions were prepared. Then 4× inhibitor stocks were preparedby adding 4 μl of an inhibitor dilution to 1 ml of RPMI1640 medium/10%fetal bovine serum. The 4× inhibitor stocks contained inhibitor atconcentrations of 80 μM, 32 μM, 12.8 μM, 5.12 μM, 2.048 μM, 0.819 μM,0.328 μM, 0.131 μM, 0.052 μM, 0.021 μM etc. The 4× inhibitor stocks werepre-warmed at 37° C. until use.

Fresh human blood buffy cells were separated from other cells in aVacutainer CPT from Becton & Dickinson (containing 4 ml blood and enoughDPBS without Mg²⁺ /Ca²⁺ to fill the tube) by centrifugation at 1500×gfor 15 min. Peripheral blood mononuclear cells (PBMCs), located on topof the gradient in the Vacutainer, were removed and washed twice withRPMI1640 medium/10% fetal bovine serum. PBMCs were collected bycentrifugation at 500×g for 10 min. The total cell number was determinedusing a Neubauer Cell Chamber and the cells were adjusted to aconcentration of 4.8×10⁶ cells/ml in cell culture medium (RPMI1640supplemented with 10% fetal bovine serum).

Alternatively, whole blood containing an anti-coagulant was useddirectly in the assay.

We placed 100 μl of cell suspension or whole blood in each well of a96-well cell culture plate. Then we added 50 μl of the 4× inhibitorstock to the cells. Finally, we added 50 μl of a lipopolysaccharide(LPS) working stock solution (16 ng/ml in cell culture medium) to give afinal concentration of 4 ng/ml LPS in the assay. The total assay volumeof the vehicle control was also adjusted to 200 μl by adding 50 μl cellculture medium. The PBMC cells or whole blood were then incubatedovernight (for 12-15 hours) at 37° C./5% CO₂ in a humidified atmosphere.

The next day the cells were mixed on a shaker for 3-5 minutes beforecentrifugation at 500×g for 5 minutes. Cell culture supernatants wereharvested and analyzed by ELISA for levels of IL-1b (R & D Systems,Quantikine kits, #DBL50), TNF-A (BioSource, #KHC3012), IL-6 (Endogen,#EH2-IL6) and IL-8 (Endogen, #EH2-IL8) according to the instructions ofthe manufacturer. The ELISA data were used to generate dose-responsecurves from which IC₅₀ values were derived.

Results in whole blood for the p38 inhibitor compound 6 are shown inTable 4 below:

    ______________________________________                                        Cytokine assayed                                                                            IC.sub.50 (whole blood; μM)                                  ______________________________________                                        IL-1          3                                                               TNF           1                                                               IL-6          1                                                               ______________________________________                                    

Results for various p38 inhibitors of this invention in theLPS-stimulation assay are shown in Table 5:

    ______________________________________                                        Compound #   IL-1 (IC.sub.50 ; μM)                                                                  TNF (IC.sub.50 ; μM)                              ______________________________________                                        2            15          20                                                   3            14          14                                                   6            0.45        0.45                                                 7            5           20                                                   8            3.5         10                                                   9            >20         >20                                                  10           20          >20                                                  11           2           >20                                                  12           0.1         0.2                                                  13           0.7         0.9                                                  14           0.35        0.87                                                 15           0.21        0.28                                                 16           0.97        2.2                                                  ______________________________________                                    

Other p38 inhibitors of this invention will also inhibit the productionof IL-1, TNF and IL-6, as well as IL-8 in LPS-stimulated PBMCs or wholeblood.

D. Inhibition of IL-6 and IL-8 Production in IL-1-Stimulated PBMCs

This assay was carried out on PBMCs exactly the same as above exceptthat 50 μl of an IL-1b working stock solution (2 ng/ml in cell culturemedium) was added to the assay instead of the (LPS) working stocksolution.

Cell culture supernatants were harvested as described above and analyzedby ELISA for levels of IL-6 (Endogen, #EH2-IL6) and IL-8 (Endogen,#EH2-IL8) according to the instructions of the manufacturer. The ELISAdata were used to generate dose-response curves from which IC₅₀ valueswere derived.

Results for p38 inhibitor compound 6 are shown in Table 5 below:

    ______________________________________                                        Cytokine assayed                                                                              IC.sub.50 (μM)                                             ______________________________________                                        IL-6            0.60                                                          IL-8            0.85                                                          ______________________________________                                    

E. Inhibition of LPS-Induced Prostaglandin Endoperoxide Synthase-2(PGHS-2, or COX-2) Induction In PBMCs

Human peripheral mononuclear cells (PBMCs) were isolated from freshhuman blood buffy coats by centrifugation in a Vacutainer CPT (Becton &Dickinson). We seeded 15×10⁶ cells in a 6-well tissue culture dishcontaining RPMI 1640 supplemented with 10% fetal bovine serum, 50U/mlpenicillin, 50 μg/ml streptomycin, and 2 mM L-glutamine. Compound 6(above) was added at 0.2, 2.0 and 20 μM final concentrations in DMSO.Then we added LPS at a final concentration of 4 ng/ml to induce enzymeexpression. The final culture volume was 10 ml/well.

After overnight incubation at 37° C., 5% CO₂, the cells were harvestedby scraping and subsequent centrifugation, then the supernatant wasremoved, and the cells were washed twice in ice-cold DPBS (Dulbecco'sphosphate buffered saline, BioWhittaker). The cells were lysed on icefor 10 min in 50 μl cold lysis buffer (20 mM Tris-HCl, PH 7.2, 150 mMNaCl, 1% Triton-X-100, 1% deoxycholic acid, 0.1% SDS, 1 mM EDTA, 2%aprotinin (Sigma), 10 μg/ml pepstatin, 10 μg/ml leupeptin, 2 mM PMSF, 1mM benzamidine, 1 mM DTT) containing 1 μl Benzonase (DNAse from Merck).The protein concentration of each sample was determined using the BCAassay (Pierce) and bovine serum albumin as a standard. Then the proteinconcentration of each sample was adjusted to 1 mg/ml with cold lysisbuffer. To 100 μl lysate an equal volume of 2×SDS PAGE loading bufferwas added and the sample was boiled for 5 min. Proteins (30 μg/lane)were size-fractionated on 4-20% SDS PAGE gradient gels (Novex) andsubsequently transferred onto nitrocellulose membrane by electrophoreticmeans for 2 hours at 100 mA in Towbin transfer buffer (25 mM Tris, 192mM glycine) containing 20% methanol. The membrane was pretreated for 1hour at room temperature with blocking buffer (5% non-fat dry milk inDPBS supplemented with 0.1% Tween-20) and washed 3 times in DPBS/0.1%Tween-20. The the membrane was incubated overnight at 4° C. with a 1:250dilution of monoclonal anti-COX-2 antibody (Transduction Laboratories)in blocking buffer. After 3 washes in DPBS/0.1% Tween-20, the membranewas incubated with a 1:1000 dilution of horseradishperoxidase-conjugated sheep antiserum to mouse Ig (Amersham) in blockingbuffer for 1 h at room temperature. Then the membrane was washed again 3times in DPBS/0.1% Tween-20 and an ECL detection system (SuperSignal™CL-HRP Substrate System, Pierce) was used to determine the levels ofexpression of COX-2.

Results of the above mentioned assay indicate that compound 6 inhibitsLPS induced PGHS-2 expression in PBMC's.

While we have hereinbefore presented a number of embodiments of thisinvention, it is apparent that our basic construction can be altered toprovide other ebodiments which utilize the methods of this invention.

We claim:
 1. A compound of the formula: ##STR38## wherein: each of Q₁and Q₂ are independently selected from phenyl or pyridyl;wherein: Q₁ issubstituted with 1 to 4 substituents, independently selected from halo;C₁ -C₃ alkyl; C₁ -C₃ alkyl substituted with NR', OR', CO₂ R' or CONR'₂ ;O--(C₁ -C₃)-alkyl; O--(C₁ -C₃)-alkyl substituted with NR', OR', CO₂ R'or CONR'₂ ; NR'₂ ; OCF₃ ; CF₃ ; NO₂ ; CO₂ R'; CONR'; SR'; SCF₃ ; or CN;and Q₂ is optionally substituted with up to 4 substituents,independently selected from halo; C₁ -C₃ alkyl; C₁ -C₃ alkyl substitutedwith NR', OR', CO₂ R' or CONR'₂ ; O--(C₁ -C₃)-alkyl; O--(C₁ -C₃)-alkylsubstituted with NR', OR', CO₂ R' or CONR'₂ ; NR'₂ ; OCF₃ ; CF₃ ; NO₂ ;CO₂ R'; CONR'; SR'; SCF₃ ; or CN; wherein R' is selected from hydrogen,(C₁ -C₃)-alkyl or (C₂ -C₃)-alkenyl or alkynyl; X is selected from --S--,--O--, --S(O₂)--, --S(O)--, --S(O₂)--N(R)--, --N (R) --S(O₂)--,--N(R)--C(O)O--, --O--C(O)--N(R)--, --C(O)--, --C(O)O--, --O--C(O)--,--C(O)--N(R)--, --N(R) --C(O)--, --N(R)--, or --C(R₂)--; each R isindependently selected from hydrogen, --R², --NR² ₂, --OR², SR²,--C(O)--NR² ₂ or --C(O)--OR² wherein two adjacent R are optionally boundto one another and, together with each Y to which they are respectivelybound, form a 4-8 membered carbocyclic or heterocyclic ring; R² isselected from hydrogen, (C₁ -C₃)-alkyl, O--(C₂ -C₃)-alkyl, (C₁-C₃)-alkenyl or O--C(C₂ -C₃)-alkenyl, each optionally substituted withW; W is selected from --N(R')₂, --OR', --SR', --S(O)R', --S(O₂)R',C(O)OR', ##STR39## or any 5 to 7-membered heterocyclic ring comprisingup to 3 heteroatoms independently selected from N, O or S, wherein saidheterocyclic ring is optionally substituted with up to 3 substituentsindependently selected from halo, C₁ -C₃ alkyl, O--(C₁ -C₃)-alkyl, NR'₂,OCF₃, CF₃, NO₂, CO₂ R', CONR' or CN; Y is C; A is N; n is 1 and R₁ isselected from hydrogen, (C₁ -C₃)-alkyl, OH, or O--(C₁ -C₃)-alkyl.
 2. Thecompound according to claim 1, wherein Q₁ is selected from phenyl orpyridyl containing 1 to 3 substituents independently selected fromchloro, fluoro, bromo, --CH₃, --OCH₃, --OH, --CF₃, --OCF₃, --O(CH₂)₂ CH₃or NH₂, and wherein at least one of said substituents is in the orthoposition and said substituents are independently selected from chloro,fluoro, bromo, --CH₃, --OCH₃, --OH, --CF₃, --OCF₃, --O(CH₂)₂ CH₃ or NH₂.3. The compound according to claim 2, wherein Q₁ contains at least twosubstituents, both of which are in the ortho position.
 4. The compoundaccording to claim 2, wherein Q₁ is selected from: ##STR40##
 5. Thecompound according to claim 3, wherein Q₁ is selected from2,6-difluorophenyl or 2,6-dichlorophenyl.
 6. The compound according toclaim 1, wherein Q₂ is selected from phenyl or pyridyl and wherein Q₂optionally contains up to 3 substituents, each of which is independentlyselected from chloro, fluoro, methyl, --OCH₃, --OH, --NH₂, --CF₃,--OCF₃, --SCH₃, --OCH₃, --C(O)OH and --CH₂ CH₂ OH.
 7. The compoundaccording to claim 6, wherein, Q₂ is selected from: or unsubstitutedphenyl.
 8. The compund according to claim 7, wherein Q₂ is selected fromphenyl or 4-fluorophenyl.
 9. The compound according to claim 1, whereinX is selected from --S--, --O--, --S(O₂)--, --S(0)--, --NR--, --C(R₂)--or --C(O)--.
 10. The compound according to claim 9, wherein X is S. 11.The compound according to claim 1, wherein each R attached to Y isindependently selected from hydrogen or methyl.
 12. The compoundaccording to claim 11, having formula: ##STR41## and being selected fromthe compounds indicated in the table below:

    ______________________________________                                        Cmpd# Q.sub.1      Q.sub.2    R.sub.a                                                                              R.sub.b                                  ______________________________________                                        2     4-fluorophenyl                                                                             phenyl     hydrogen                                                                             hydrogen                                 3     2,4-dichlorophenyl                                                                         phenyl     hydrogen                                                                             hydrogen                                 5     2,4-dichlorophenyl                                                                         4-methylphenyl                                                                           hydrogen                                                                             hydrogen                                 6     2,6-dichlorophenyl                                                                         phenyl     hydrogen                                                                             hydrogen                                 7     2-chlorophenyl                                                                             phenyl     hydrogen                                                                             hydrogen                                 8     2-methylphenyl                                                                             phenyl     hydrogen                                                                             hydrogen                                 9     3,4-dichlorophenyl                                                                         phenyl     hydrogen                                                                             hydrogen                                 10    4-methoxyphenyl                                                                            phenyl     hydrogen                                                                             hydrogen                                 11    2-methoxyphenyl                                                                            phenyl     hydrogen                                                                             hydrogen                                 12    2,6-dichlorophenyl                                                                         4-fluorophenyl                                                                           hydrogen                                                                             hydrogen                                 13    2,6-dichlorophenyl                                                                         phenyl     methyl methyl                                   14    2,6-dichlorophenyl                                                                         4-methylphenyl                                                                           hydrogen                                                                             hydrogen                                 15    2,6-dichlorophenyl                                                                         3-methlyphenyl                                                                           hydrogen                                                                             hydrogen                                 16    2,6-dichlorophenyl                                                                         3,4-       hydrogen                                                                             hydrogen.                                                   dichlorophenyl                                             ______________________________________                                    


13. A pharmaceutical composition comprising an amount of a compoundaccording to any one of claims 1 to 12 effective to inhibit p 38, and apharmaceutcially acceptable carrier.
 14. A method of treating orpreventing inflammatory disease, autoimmune disease, destructive bonedisorder, proliferative disorder, infectious disease, viral disease, orneurodegenerative disease in a pateint, said method comprisingadministering to said patient a composition according to claim
 13. 15.The method according to claim 14, wherein said method is used to treator prevent an inflammatory disease selected from acute pancreatitis,chronic pancreatitis, asthma, allergies, or adult respiratory distresssyndrome.
 16. The method according to claim 14, wherein said method isused to treat or prevent an autoimmune disease selected fromglomeralonephritis, rheumatoid arthritis, systemic lupus erythematosus,scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis,insulin-dependent diabetes mellitus (Type I), autoimmune hemolyticanemia, autoimmune neutropenia, thrombocytopenia, atopic dermatitis,chronic active hepatitis, myasthenia gravis, multiple sclerosis,inflammatory bowel disease, ulcerative colitis, Crohn's disease,psoriasis, or graft vs. host disease.
 17. The method according to claim14, wherein said method is used to treat or prevent a destructive bonedisorders selected from osteoarthritis, osteoporosis or multiplemyeloma-related bone disorder.
 18. The method according to claim 14,wherein said method is used to treat or prevent a proliferative diseaseselected from acute myelogenous leukemia, chronic myelogenous leukemia,metastatic melanoma, Kaposi's sarcoma, or multiple myeloma.
 19. Themethod according to claim 14, wherein said method is used to treat orprevent an infectious disease selected from sepsis, septic shock, orShigellosis.
 20. The method according to claim 14, wherein said methodis used to treat or prevent a viral disease selected from acutehepatitis infection, HIV infection or CMV retinitis.
 21. The methodaccording to claim 14, wherein said method is used to treat or prevent aneurodegenerative disease selected from Alzheimer's disease, Parkinson'sdisease or cerebral ischemia.
 22. A method of treating or preventingischemia/reperfusion in stroke, or myocardial ischemia, renal ischemia,heart attacks, organ hypoxia or thrombin-induced platelet aggregation ina patient, said method comprising the step of administering to saidpatient a pharmaceutical composition according to claim
 12. 23. A methodof inhibiting prostaglandin endoperoxide synthase-2 in a patient,comprising the step of administering to said patient a pharmaceuticalcomposition according to claim
 12. 24. The method according to claim 23,wherein said method is used to treat or prevent edema, fever, analgesiaor to manage pain.
 25. The method according to claim 24, wherein saidpain is selected from neuromuscular pain, headache, cancer pain, dentalpain or arthritis pain.